Antiproliferation drugs, e.g. anti-tumor or anticancer drugs, and their analogues, derivatives and prodrugs are well known for use in the treatment of cancer, tumor and other cellular proliferation related diseases. Methods for their administration are varied and have met with a range of successes and failures. Goals in the development of antiproliferative agent-containing pharmaceutical compositions and formulations include improving targeted delivery of these drugs in order to minimize systemic toxicity to subjects being treated with these drugs, maintaining efficacy of these drugs upon derivatization thereof. There have been some successes in improving targeted delivery of the drugs to cancer or tumor cells, and maintaining the efficacy of the drugs even after derivatization thereof. Even so, targeted delivery combined with high efficacy remains a key goal in anti-tumor therapy.
Derivatization of drugs with naturally occurring biologically active components has been evaluated as a means for improving targeted delivery of these drugs. To that end, cobalamin conjugates comprising drugs, proteins, nucleic acids, amino acids, peptides, hormones or other components have been developed in an effort to improve bioavailability by exploiting the biological mechanism of cobalamin cellular uptake. In principle, a bioconjugate (CBD) comprising an agent covalently linked to cobalamin (CB) becomes bound to a protein in vivo. Depending upon the site of administration, the protein is Intrinsic Factor (IF) or transcobalamin (TCCB) I, II or III. IF is a naturally occurring protein in the gastrointestinal tract that binds to CB. After oral administration of a cobalamin bioconjugate, it is bound to IF to form a complex (IF-CBD) that is actively transported across the lumen of the GI tract. Once in the plasma, IF dissociates from the IF-CBD complex thereby releasing CBD into the plasma compartment. TCCB is responsible for cellular uptake of CB across the cellular membrane. TCCB binds to CBD in the plasma to form a complex TCCB-CBD. The TCCB-CBD complex is then actively transported across the cellular membrane via the TCCB receptors on the cell membrane. After entry into the cell, TCCB undergoes intracellular dissociation from the TCCB-CBD complex thereby releasing CBD intracellularly. A number of publications report the preparation of cobalamin conjugates for the above-mentioned uses.
U.S. Pat. Nos. 5,739,313; 6,004,533; 6,096,290; 6,211,355; and PCT Publication WO 97/18231 disclose radionuclide labeling of vitamin B12 through the propionamide moieties on naturally occurring vitamin B12. The propionamide moieties at the b-, d-, and e-positions of the corrin ring were converted to monocarboxylic acids, through a mild hydrolysis, and the carboxylic acids were separated by column chromatography. A bifunctional linking moiety was then attached to the carboxylate function through an amide linkage, and a chelating agent was attached to the linking moiety again through an amide linkage. The chelating moiety was used to attach a radionuclide to the vitamin so that it could be used for therapeutic or diagnostic purposes.
Hogenkamp et al. in WO 01/28595 (PCT/USOO/10098) disclose a series of cobalamin conjugates that are linked via a protein linker to a detectable group, which is useful in the imaging of tumors. The linker is not attached to cobalamin via the 5′-OH group. Hogenkamp et al. suggest that such compounds may be useful in the treatment of tumors.
Hogenkamp et al. in WO 01/28592 (PCT/USOO/10097) disclose a series of cobalamin conjugates that are linked directly to or indirectly by a linker to a residue of a chemotherapeutic agent, which is useful in the treatment of abnormal cellular proliferation. The linker is not attached to cobalamin via the 5′-OH group. A doxorubicin conjugate is contemplated and a proposed synthesis therefor is included in the application. No actual exemplification of compounds is included in the application even though a wide range of antiproliferative agents are disclosed as being suitable. The conjugation is proposed to occur through a carboxylic acid moiety of the cobalamin.
Collins et al. in WO 00/62808 (PCT/USOO/10100) disclose a series of cobalamin conjugates that are linked directly or by a linker to a residue of a molecule comprising B-10 or Gd-157, which are useful in the treatment of abnormal cellular proliferation. A neutron capture therapy target is linked to cobalamin. After systemic administration, the conjugate is absorbed into a tumor cell. Then neutron capture irradiation is administered. Proposed linkage sites include the amide sites and the cobalt. The 5′-OH of the cobalamin ribose ring is not proposed.
PCT Publication WO 98/08859 to Grissom et al. discloses conjugates containing a bioactive agent and an organocobalt complex in which the bioactive agent is covalently bound directly or indirectly, via a spacer, to the cobalt atom. The bioactive agent can be a chemotherapeutic agent (anti-tumor drug). The bioactive agent is released from the bioconjugate by the cleavage of the weak covalent bond between the bioactive agent and the cobalt atom as a result of normal displacement by cellular nucleophiles or enzymatic action, or by application of an external signal (e.g., light, photoexcitation, ultrasound, or the presence of a magnetic field). The conjugates are reportedly targeted for site specific release of bioactive agents in cells, tissues, or organs.
PCT International Publication WO 03/025139 to Collins et al. discloses a conjugate for the delivery of nucleic acids via coupling to VB12, cobalamin. A degradable linker is used. Proposed linkage is through any of the amide sites or the 5′-OH site. Antisense sequences, nonsense sequences, antisense mimics, nucleic acids and nucleic acid analogues are contemplated.
U.S. Pat. No. 5,428,023 to Russell-Jones et al. discloses a vitamin B12 conjugate for delivering oral hormone formulations. Russell-Jones teaches that the vitamin B12 conjugate must be capable of binding in vivo to intrinsic factor, enabling uptake and transport of the complex from the intestinal lumen of a vertebrate host to the systemic circulation of the host. The hormones are attached to the vitamin B12 through a hydrolyzed propionamide linkage on the vitamin. The patent states that the method is useful for orally administering hormones, bioactive peptides, therapeutic agents, antigens, and haptens, and lists as therapeutic agents neomycin, salbutamol cloridine, pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine, xylazine, ketamine hydrochloride, mephanesin and iron dextran.
U.S. Pat. No. 5,548,064 and No. 6,262,253 to Russell-Jones et al. disclose a vitamin B12 conjugate for delivering erythropoietin and granulocyte colony stimulating factor, using the same approach as the '023 patent.
PCT Publication WO 94/27641 to Russell-Jones et al discloses vitamin B12 linked through a polymer to various active agents wherein the conjugate is capable of binding to intrinsic factor for systemic delivery. In particular, the document discloses the attachment of various polymeric linkers to the propionamide positions of the vitamin B12 molecule, and the attachment of various bioactive agents to the polymeric linker. Exemplary bioactive agents include hormones, bioactive peptides and polypeptides, anti-tumor agents, antibiotics, antipyretics, analgesics, anti-inflammatory agents, and haemostatic agents. Exemplary polymers include carbohydrates and branched chain amino acid polymers. The linkers used in WO 94/27641 are polymeric (each having a molecular weight of about 5000 or greater). The linkers are described as exhibiting a mixture of molecular weights, due to the polymerization process by which they are made.
PCT Publication WO 99/65930 and U.S. Pat. No. 6,150,341 to Russell-Jones et al. disclose the attachment of various agents to the 5′-OH position on the vitamin B12 (VB12) ribose ring. The publications indicate that the system can be used to attach polymers, nanoparticles, therapeutic agents, proteins, and peptides to the vitamin. Russell-Jones et al. disclose the preparation of 5′-OH VB12 derivatives via the use of an active carbonyl electrophile. After reacting the VB12 5′-OH with the carbonyl electrophile, a linker, diamino spacer, or other molecule is reacted with activated 5′-OH site. Alternatively, the 5′-OH site is converted to an ester site that is then derivatized. Linking of the VB12 derivatives to drugs is contemplated.
PCT International Publication WO 02/074171 (PCT/US02/08285) and U.S. Pregrant Patent Publication 2002/0192683 to Grissom et al. disclose fluorescent cobalamin derivatives. A fluorescent moiety is linked to the cobalamin preferably via the corrin ring or the 5′-OH group. The compounds are used to detect cancer cells, identify cells that are potentially susceptible to anticancer therapy and other such methods. It does not disclose an anticancer drug attached to the 5′-OH group.
U.S. Pat. No. 5,574,018 to Habberfield et al. discloses conjugates of vitamin B12 in which a therapeutically useful protein is attached to the primary hydroxyl site of the ribose moiety. The patent lists erythropoietin, granulocyte-colony stimulating factor and human intrinsic factor as therapeutically useful proteins, and indicates that the conjugates are particularly well adapted for oral administration.
U.S. Pat. No. 5,840,880 to Morgan, Jr. et al. discloses vitamin B12 conjugates to which are linked receptor modulating agents, which affect receptor trafficking pathways that govern the cellular uptake and metabolism of vitamin B12. The receptor modulating agents are linked to the vitamin at the b-, d-, or e-position.
U.S. Pregrant Patent Publication 2002/0151525 to Collins et al. discloses a range of conjugates of VB-12 linked to an antiproliferative drug. The drug can be linked by a variety of different linkers at a number of different sites on the VB-12 molecule including the 5′-OH site. Doxorubicin is included among a laundry list of suitable antiproliferative drugs. Although a prophetic and general description for synthesis of a conjugate comprising doxorubicin attached to a carboxylic acid moiety of VB-12 is disclosed, there is no actual exemplification of such compound. There is also no other synthetic procedure disclosed for any other specific conjugates, especially conjugates of the 5′-OH site.
U.S. Pregrant Patent Publications No. 2002/0115595 and No. 2002/0049154 to Grissom et al. discloses organocobalt derivatives of VB12. The derivatives are disclosed as being suitable for oral and i.v. administration. Several anti-tumor drugs, such as doxorubicin, methotrexate, and carboplatin, are disclosed as being suitable for conjugation. Cleavage of the anti-tumor drug from a self-destructing linker is proposed to occur by cellular nucleophiles, enzymes, light or sound. Grissom et al. propose a method of treating cancer with the conjugate.
U.S. Pat. No. 6,315,978 to Grissom et al. discloses organocobalt derivatives of VB-12 adapted to oral or i.v. administration. Anti-tumor drugs that can be conjugated to the VB-12 include doxorubicin, methotrexate, and carboplatin. They suggest cleavage of the anti-tumor drug from the linker by cellular nucleophiles or enzymes or light or sound. The linker is a self-destructing linker that breaks away from the drug after it has been removed from the VB-12. They also suggest a method of treating cancer with the conjugate.
U.S. Pregrant Patent Publication 2002/0042394 to Hogenkamp et al. discloses VB-12 conjugated with an antibiotic optionally for use as an imaging agent. Hogenkamp identify topical antineoplasts (EFUDEX: fluorouracil; fluoroplex) as antibiotic compounds suitable for use in the conjugate. They suggest antibiotic compounds attached to VB-12 at a variety of sites including the 5′-OH. They do not disclose intravenous administrable anti-tumor conjugates.
U.S. Pat. No. 5,449,720 to Russell-Jones et al. discloses the use of polymer as a linker between VB-12 and an active agent. The conjugate is defined as (V-Q)n-P-(Q′-A)m where V is VB-12; P is an optionally biodegradable polymer; A is an active agent; and Q, Q′ are optional spacers or cross-linking agents. Russell-Jones et al. disclose a list of potential anti-tumor agents and many other drugs that can be conjugated to the VB-12 via carboxyl moieties. However, they do not disclose 5′-OH derivatives of VB-12.
U.S. Pat. No. 5,589,463 to Russell Jones discloses utilization of the VB-12 uptake mechanism for transport of VB-12 derivatives across the lumen of the GI tract following oral administration. The VB-12 derivative linked to an active agent. A range of active agents but not anti-tumor compounds is disclosed. A cross-linking agent is used to form the linker. Antibiotics attached to VB-12 are disclosed to enhance uptake of drug. They also do not disclose 5′-OH derivatives of VB-12.
U.S. Pat. No. 5,739,287 to Wilbur et al., No. 5,840,712 to Morgan Jr. et al., No. 5,840,880 to Morgan Jr. et al., No. 5,869,465 to Morgan Jr. et al. and No. 6,083,926 to Morgan Jr. et al. disclose biotinylated VB-12 discloses designed to block VB-12 receptors. Their 5′-OH derivatives are prepared according to procedure of Toraya (Bioinorg. Chem. (1975), 4, 245-255). They disclose variety of different groups that can be used to attach the linker to the VB-12. For the treatment of cancer, they disclose coadministration of methotrexate, or another anticancer or anti-tumor drug, along with a modified VB-12 thereby employing two different mechanisms: depletion of VB-12 in growing cancer cells coupled with administration of a chemotherapeutic agent. They do not suggest conjugation of the methotrexate with the VB-12.
U.S. Pat. No. 5,807,832 to Russell-Jones et al discloses the use of a cross-linking agent to conjugate VB-12 and a bioactive molecule (hormone, antibiotic, hapten, antigen, protein, secretory product). They do not disclose intracellular enzyme cleavage of the conjugate, nor do they disclose anti-tumor drugs or 5′-OH derivatives of VB-12.
U.S. Pat. No. 5,863,900 to Russell-Jones et al. discloses LHRH antagonists (ANTIDE-1, ANTIDE-2, ANTIDE-3) linked to VB-12 via carboxylate linkage with a diamine or dithiol linkage. ANTIDE components resist enzymatic hydrolysis in the GI tract. They suggest cleavage of ANTIDE from VB-12 in vivo. They suggest the use of analogues for in vivo cleavage by transglutaminase, but did not succeed in doing so. They also do not disclose anti-tumor compounds as conjugates of VB-12.
U.S. Pat. No. 6,214,345 to Firestone et al. discloses the preparation and use of conjugates of an anti-tumor drug and a targeting ligand (antibody or protein) linked by way of a self-destructing (self-immolative spacer). VB-12 is not disclosed as a suitable ligand. Instead, macromolecules such as antibodies and the like are disclosed as suitable ligands. Doxorubicin is claimed as a drug that can be included in the conjugate.
Other patent describing the use of Vitamin B12 include U.S. Pat. No. 3,936,440 to Nath (Method of Labeling Complex Metal Chelates with Radioactive Metal Isotopes); U.S. Pat. No. 4,209,614 to Bernstein et al., (Vitamin B12 Derivatives Suitable for Radiolabeling); U.S. Pat. No. 4,279,859 (Simultaneous Radioassay of Folate and Vitamin B12); U.S. Pat. No. 4,283,342 to Yollees (Anticancer Agents and Methods of Manufacture); U.S. Pat. No. 4,301,140 to Frank et al (Radiopharmaceutical Method for Monitoring Kidneys); U.S. Pat. No. 4,465,775 to Houts (Vitamin Brand labeled Derivatives for Such Assay); U.S. Pat. No. 5,308,606 to Wilson et al (Method of Treating and/or Diagnosing Soft Tissue Tumors); U.S. Pat. No. 5,405,839 (Vitamin B, Derivative, Preparation Process Thereof, and Use Thereof); U.S. Pat. No. 5,608,060 to Axworthy et al (Biotimidase-Resistant Biotin-DOTA Conjugates); U.S. Pat. No. 5,869,465 to Morgan et al (Method of Receptor Modulation and Uses Therefor); U.S. Pat. No. 5,869,466 to Russell-Jones et al (vitamin B12 Mediated Oral Delivery systems for GCSF). See also Ruma Banerjee, Chemistry and Biochen2istry of B12 John Wiley & Sons, Inc. (1999), and in particular Part II, Section 15 of that book, entitled “Diagnostics and Therapeutic Analogues of Cobalamin,” by H. P. C. Hogenkamp, Douglas A. Collins, Charles B. Grissom, and Frederick G. West.
A conjugate comprising a porphyrin-like moiety linked to an anti-tumor drug by way of a cleavable linker has been disclosed by Han (U.S. Pregrant Publication No. 2002/0155999). Protoporphyrin is derivatized with a linker and subsequently reacted with an available functional group of an anti-tumor compound to form the conjugate. The conjugate of Han, however, is adapted for cleavage in the physiological condition surrounding the tumor rather than within the tumor.
While in vivo efficacy is the hallmark of success in anticancer and anti-tumor therapy, efficacy should not come at the cost of excessive systemic toxicity. In fact, it is highly desirable, although frequently untenable, to provide an anticancer or anti-tumor agent possessing increased toxicity toward cancer and tumor cells but decreased systemic toxicity toward the host or subject receiving the agent. A preferred anticancer or anti-tumor agent is one that provides a high kill rate for cancer or tumor cells and a low death rate for the host. The prior art does not disclose or suggest VB12 conjugates possessing reduced systemic toxicity and enhanced efficacy as compared to their corresponding free drugs, yet a need for such conjugates remains.
Accordingly, while the prior art recognizes the potential utility of cobalamin derivatives for the treatment of cancer or tumors, it has not successfully prepared antiproliferative drug 5′-OH conjugates of cobalamin, wherein the drug is attached to the cobalamin by way of a linker that is degradable or hydrolysable with an intracellular enzyme. In particular, the prior art does not disclose the preparation of such a conjugate comprising doxorubicin nor the use of a cobalamin 5′-OH-doxorubicin conjugate for the treatment of tumors or cancer.